A Novel Mutation of ADAMTS13 Gene in Congenital Thrombotic Thrombocytopenic Purpura

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4674-4674
Author(s):  
Xia Bai ◽  
Jian Su ◽  
Minghua Jiang ◽  
Zhaoyue Wang ◽  
Changgeng Ruan
Keyword(s):  
Conflicts Of Interest ◽  
Novel Mutation ◽  
Gene Mutations ◽  
Type I ◽  
Adamts13 Activity ◽  
Intron Boundary ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Peripheral Leukocytes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 4674 Congenital thrombotic thrombocytopenic purura (TTP) is caused by gene mutations of von Willebrand factor-cleaving protease (a disintegrin and metalloprotease with thrombospondin type I domains 13, ADAMTS13). In this study, one novel mutation in the ADAMTS13 gene was found in a woman whose presents are first cousins. Thrombocytopenia occurred during the second trimesters in her first pregnancy, and she died of recurrent attacks after diagnosis of TTP. The ADAMTS13 activity measured using the recombinant FRET-VWF73 during her acute episode was less than 5%. ADAMTS13 inhibitor was negative measured by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. The 29 exons and exon-intron boundary sites of ADAMTS13 gene was analyzed using the human genomic DNA extracted from peripheral leukocytes of the patient. The results demonstrated she was homozygous for R498C. This novel mutant was constructed using the expression plasmid pSectag containing ADAMTS13 cDNA, and the vector was introduced by linpofectamine 2000 to Hela cells. Western Blot revealed that rADAMTS13-wide type (WT) was synthesized as a single band with molecular mass close to 190 Kda in the conditioned media, however, no detectable ADAMTS13 of this mutant existed. The lysates of cells expressing the mutant showed the same protein amounts compared to the rADAMTS13-WT. The immunofluorescence study demonstrated that mutant had the same localization pattern at Endoplasmic Reticulum(ER)and Golgi-compartments compared to the rADAMTS13-WT. The results imply that this mutant may be retained in the cellular ER and Golgi-comparments, but rapidly degraded or insufficiently secreted. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 In 4 Different VWF/VIII Concentrates and Its Impact on Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3677-3677
Author(s):  
Mirjeta Qorraj ◽  
Tanja Falter ◽  
Sarah Steinemann ◽  
Thomas Vigh ◽  
Inge Scharrer
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Relative Reduction ◽  
Hemorrhagic Diathesis ◽  
Adamts13 Activity ◽  
Kinetic Measurements ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Anti-β2-GPI Antibodies Induce Von Willebrand Factor Release, Modulate Platelet Binding, and Affect VWF Proteolysis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1444-1444
Author(s):  
Christopher J. Ng ◽  
Keith R. McCrae ◽  
Junmei Chen ◽  
Michael Wang ◽  
Marilyn J. Manco-Johnson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Syndrome ◽  
Von Willebrand Factor ◽  
Image Acquisition ◽  
Type I ◽  
Adamts13 Activity ◽  
Normal Plasma ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: The antiphospholipid syndrome (APS) is characterized by predisposition to thrombosis. The cause for this pathology is poorly understood but is likely multifactorial, involving activation of blood cells and vasculature. The role that anti-β2-GPI antibodies play in von Willebrand factor (VWF) release from endothelial cells, VWF-platelet binding, and VWF cleavage by ADAMTS13 has not been well characterized in APS. We decided to study the effect of these antibodies on expressed ultra large VWF strings (ULVWF strings) that bind platelets (VWF-PLT strings) under flow to better understand platelet–VWF binding and ADAMTS13 regulation in APS. Hypothesis: We hypothesized that Anti-β2-GPI antibodies could induce VWF release from endothelial cells and modulate VWF’s prothombotic effect through alterations in VWF-Platelet binding and VWF cleavage by ADAMTS13. Methods: Human umbilical vein endothelial cells were seeded in 96-well plates/flow chambers prepared with a collagen Type I substrate for static/flow experiments, respectively. Static assays: Cells were incubated for 1 hr with Anti-β2-GPI or control antibodies and the conditioned media was assayed for VWF by ELISA, normalized to normal plasma. Flow Assay Analysis: After stimulation with agonist and perfusion with a platelet suspension, platelets bound to ULVWF in a string pattern were quantified via brightfield microscopy. Images of chambers were captured and VWF-PLT string-units (defined as a string length of 25μM) per slide were quantified. To minimize bias, image acquisition was standardized and the investigator was blinded at time of image acquisition/analysis. β2-GPI Flow assays: Endothelial cells in flow chambers were stimulated with 50ng/mL of phorbol myristate acetate (PMA), and a solution of fixed platelets with β2-GPI or β2-GPI+Anti-β2-GPI were perfused prior to image acquisition. ADAMTS13 assays: After stimulation with 25ng/mL PMA and perfusion with fixed platelets, images were acquired. Then control/patient plasma was perfused over formed strings. Images taken after plasma perfusion were quantified and compared to images prior to plasma perfusion. Data are shown as mean +/- SEM, and significance was determined as p<0.05 by student’s t-test or Mann-Whitney U Test, when appropriate. Results: Static Assays: Compared to control human IgG (8.28 +/- 3.34 mU/mL), VWF release was increased in the presence of two patient-derived Anti-β2-GPI antibodies, APS25-6 Anti-β2-GPI, 35.73 +/- 7.83 mU/mL (P = 0.008) and APS203-2 Anti-β2-GPI, 34.08 +/- 7.119 mU/mL (P = 0.039). As compared to control rabbit IgG (15.80 +/- 7.12 mU/mL), a rabbit polyclonal Anti-β2-GPI antibody, R24-6, also demonstrated increased soluble VWF (43.16 +/- 9.60 mU/mL, P = 0.013) release. β2GPI Flow Assays:The presence of β2GPI (2µM) reduced String-unit formation from 50.10 +/-5.57 Sting-units/image to 20.98 +/- 2.05 String Units/image (P < 0.0001) as compared to buffer. Addition of goat Anti-β2-GPI antibody (1µM) increased the VWF-PLT string observed as compared to β2GPI (2µM), 30.09 +/- 1.83 String Units to 20.98 +/- 2.05 String Units (P = 0.012) indicating that an Anti-β2-GPI antibody partially reverses the effect of β2GPI on reducing VWF-PLT string formation. ADAMTS13 Assay:Compared to pooled normal plasma (ADAMTS13 Activity 100%) (4.57 +/- 0.60 String Units/image cleaved), there was a significant decrease in the amount of string units/image cleaved in two APS plasmas with Anti-β2-GPI antibodies, APS232-9 (-0.23 +/- 0.98, P = 0.0003) and APS227-9 (2.23 +/- 0.73, P = 0.0009). ADAMTS13 Activity of patient plasma was 98.37% and 83.97%, respectively. These results suggest an inhibitory role of APS plasma on the cleavage of ULVWF strings. Conclusions: Anti-β2-GPI antibodies and antiphospholipid syndrome plasma may contribute to the prothrombotic phenotype observed in APS by three mechanisms: 1) the increased release of VWF from endothelial cells after incubation with Anti-β2-GPI, 2) increased platelet binding to ULVWF strings likely mediated by interfering with β2GPI’s known inhibition of Gp1bα VWF-platelet binding, and 3) a reduced ability to cleave VWF-PLT strings by APS plasma, suggestive of ADAMTS13 inhibition that does not correlate with ADAMTS13 activity. Taken together, our results suggest that VWF and its modulation may contribute to the prothrombotic phenotype observed in the antiphospholipid syndrome. Disclosures No relevant conflicts of interest to declare.

Three Novel ADAMTS-13 Gene Mutations in Thrombotic Thrombocytopenic Purpura (TTP).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3965-3965
Author(s):  
Nam Keun Kim ◽  
Moon Ju Jang ◽  
Sun Kim ◽  
Sun Ju Lee ◽  
Hye Won Park ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Thrombus Formation ◽  
Gene Mutations ◽  
Missense Mutations ◽  
Thrombocytopenic Purpura ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Splicing Site

Abstract ADAMTS-13 is an enzyme which cleave von Willebrand factor (VWF) to prevent excessive platelet thrombus formation. Mutation of ADAMTS-13 is associated with congenital thrombotic thrombocytopenic purpura (TTP). In this study, we report three novel missense mutations of ADAMTS-13 gene in TTP families. Genetic analysis of ADAMTS-13 gene was performed in 6 TTP families. Three novel ADAMTS-13 gene mutations (2708C>T [S903L], 3650T>C [I1217T] and 3941C>T [S1314L]) detected in 3 families. They were all found with heterozygous genotype in exon 21, 26 and 28, respectively. The 3650T>C mutation was found at exon 26 in the patient and his mother. A heterozygous guanine to adenine substitution was found at 5′ splicing site of intron 3(IVS+ 1) in the patient, his brother and his father. The plasma ADAMTS-13 activities of the patient, father, mother, and brother were less than 3%, 56%, 55% and 62% respectively. ADAMTS-13 inhibitors were not detected in all family members. 3941C>T was found at exon 28 in the patient and her father. The plasma ADAMTS-13 activities of the patient, father, and mother were 96%, 83%, and 83% respectively.

scholarly journals Characterization of Two Cases of Congenital Thrombotic Thrombocytopenic Purpura (TTP)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5005-5005
Author(s):  
Xia Bai ◽  
JIan Su ◽  
Lijuan Cao ◽  
Changgeng Ruan
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Binding Assay ◽  
Conflicts Of Interest ◽  
Hek293 Cells ◽  
Novel Mutations ◽  
Thrombocytopenic Purpura ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Introduction: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by disseminated thrombus due to the mutations of ADAMTS13, which cleaves its substrate von Willebrand factor(VWF) in shear-induced unfolding condition. Most of the congenital TTP we found is woman with pregnancy. Here, we characterize two children suspected with congenital TTP. Methods:ADAMTS13 activities were analyzed by residual collagen binding assay (R-CBA) plus FRET-VWF substrate. And the inhibitors of ADAMTS13 were analyzed by 9:1 mixture of patient and pooled normal plasma followed by R-CBA. The secretion of recombinant ADAMTS13 mutants was studied. Results: Two children, one aged four months and the other aged three years old, were diagnosed with congenital TTP because their ADAMTS13 activities were less than 5% (both FRET-VWF and R-CBA), and there are short of ADAMTS13 inhibitors. The following mutations were found: Q1385P, Y177C, C522R. In addition to the previously reported mutation of Y177C, the two novel mutations (Q1385P and C522R) failed to secrete from the HEK293 cells. Conclusion: Two children with congenital TTP were determined thanks to screening of ADAMTS13 activity and its corresponding inhibitor assays, and it seems that congenital TTP could occur in different ages although most of congenital TTP we found were women with pregnancy. Disclosures No relevant conflicts of interest to declare.

Evaluation and Clinical Application of a New Method for Detecting ADAMTS13 Activity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2134-2134
Author(s):  
Anyou Wang ◽  
Ningzheng Dong ◽  
Zhenni Ma ◽  
Jingyu Zhang ◽  
Jian Su ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Sandwich Elisa ◽  
Correlation Factor ◽  
Adamts13 Activity ◽  
Simple Method ◽  
Severe Deficiency ◽  
Before And After ◽  
Von Willebrand ◽  
Relationship Of ◽  
Willebrand Factor

Abstract Abstract 2134 Poster Board II-111 Introduction: A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, and not appropriate for routine laboratories. ADAMTS13 cleaves the von Willebrand factor (VWF) within the domain A2, which locates between the domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structure of VWF before and after the cleavage. Materials and Methods: To determine the activity of ADAMTS13, a new simple method has been developed in this study. Firstly, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies (SZ-129 and SZ-125), which are specifically recognize the VWF A1 and A3 domain, respectively, by using a two-site sandwich ELISA. Comparing with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibodies in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated. Results Our results showed that plasma ADAMTS13 activities determined by the new assay were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays, and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activities of ADAMTS13 autoantibodies were ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient. Conclusions This new and simple assay for ADAMTS13 activity could be used routinely in clinic to determine the activity of ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Normal Cleavage of Von Willebrand Factor by ADAMTS13 In the Absence of Factor VIII In Patients with Severe Hemophilia A

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2114-2114
Author(s):  
Junmei Chen ◽  
Dominic W. Chung ◽  
Jennie Le ◽  
Barbara A. Konkle ◽  
José A. López
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Adamts13 Activity ◽  
Severe Hemophilia ◽  
Post Infusion ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2114 The size of von Willebrand factor (VWF), a carrier protein for factor VIII (FVIII), is regulated by plasma metalloprotease ADAMTS13 proteolytic activity. Recently studies by Cao et al. (PNAS, 2008; 105: 7416–7421) found that under shear stress, exogenous FVIII enhanced ADAMTS13 cleavage of VWF, especially the high molecular weight multimers, in a system using purified proteins. Based on this result, the authors suggested that in the absence of FVIII, such as in patients with severe hemophilia A, VWF will have ultra-large multimers due to defects in ADAMTS13 proteolytic process, which can be corrected by infusion of FVIII. Here, we assessed VWF multimers, antigen, and ADAMTS13 activity in citrated plasma from seven patients with severe hemophilia A. The FVIII levels in six patients were less than 1% and in one was 4%. Plasma from two patients was available both pre- and post-FVIII replacement therapy (recombinant FVIII). All patients displayed VWF multimer patterns similar to those in pooled normal plasma (PNP), and the two patients receiving FVIII infusions displayed no change in VWF multimer size or pattern between their pre- and post-infusion samples. In all patients, the VWF antigen level (0.32–0.76) was below the PNP value (designated as 1), and all had increased ADAMTS13 activity (measured by the ability of plasma to cleave a small A2 peptide substrate) (1.09–1.79, PNP designated as 1), yielding an increased ratio of ADAMTS13 activity to VWF antigen in these patients (1.4–5.2 compared to PNP). We also examined cleavage of endogenous VWF by ADAMTS13 in the plasmas of the two patients studied pre- and post-infusion, yielding different FVIII levels. In this assay, we first diluted plasma 10-fold with a buffer containing 10 mM HEPES, 6.5 mM BaCl2, and 1.5 M urea, incubated at 37°C, and ADAMTS13 cleavage was stopped at different time points with EDTA. VWF multimer patterns were examined on a 1.5% agarose gel. We found that ADAMTS13 cleaved VWF efficiently in patient plasma deficient in FVIII, similar to that in PNP. The extent of cleavage was correlated with the ratio of ADAMTS13 activity to VWF antigen, rather than with the FVIII levels. In conclusion, patients with severe hemophilia A appear to have normal ADAMTS13 processing of VWF multimers in vivo and ex vivo. Further studies of the effect of FVIII and VWF levels on ADAMTS13 cleavage of VWF and clinical correlation are needed. Disclosures: No relevant conflicts of interest to declare.

Pregnancies Complicated By Severe Preeclampsia Exhibit Perturbed Von Willebrand Factor (VWF)-Associated Parameters

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3525-3525 ◽  
Author(s):  
Danielle N. Drury-Stewart ◽  
Kara K. Hoppe ◽  
Kerry W. Lannert ◽  
Dominic W. Chung ◽  
Hilary S. Gammill ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Severe Preeclampsia ◽  
Adamts13 Activity ◽  
Healthy Pregnancy ◽  
Elevated Liver Enzymes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background Elevated levels of the critical coagulation glycoproteins von Willebrand Factor (VWF) and Factor VIII (FVIII) are associated with thrombovascular disease. VWF and FVIII levels are elevated in healthy pregnancy compared with the nonpregnant state, and further increases have been reported in pregnancies complicated by preeclampsia, a potentially life-threatening vascular disorder of pregnancy. Aside from the quantitative increases in VWF and FVIII, data is inconsistent or little is known regarding other key VWF-associated parameters in pregnancies complicated by preeclampsia. Hypothesis We hypothesized that pregnancies complicated by severe preeclampsia would exhibit distinct changes in VWF-associated parameters compared to healthy pregnancies. Methods We assayed VWF-associated parameters in subjects newly diagnosed with severe preeclampsia, based on standard blood pressure (BP) and proteinuria criteria. Severe preeclampsia was defined on the basis of one or more of the following: severe hypertension (systolic BP >160 or diastolic BP >110) for at least 6 hours, seizures (eclampsia), hemolysis, elevated liver enzymes, thrombocytopenia, pulmonary edema, renal dysfunction, or fetal growth restriction. We compared results from preeclamptic pregnancies to gestational age-matched healthy pregnant controls. VWF antigen (VWF:Ag), VWF propeptide (VWF:pp), and FVIII antigen (FVIII:Ag) levels were determined by ELISA. Factor VIII activity (FVIII:C) was measured using a one-stage assay and ADAMTS13 activity was determined via cleavage of an enzyme-linked peptide substrate. Results Forty-two patients with severe preeclampsia and a cohort of thirty-nine controls of similar gestational ages met study inclusion criteria (Table 1). The results of the coagulation assays are presented in Table 2. In summary, preeclamptic pregnancies were notable for statistically significant (p<0.001) increases in VWF:Ag (1.4-fold), VWF:pp (1.5-fold), and FVIII:Ag (1.2-fold) compared to controls. FVIII:Ag and FVIII:C were similar, and FVIII:Ag was used in the calculation of the VWF:FVIII ratio. The VWF:FVIII ratio trended higher in preeclamptic pregnancies, but this did not reach statistical significance (p=0.058). The mean ADAMTS13 activity in patients with preeclampsia was also significantly lower (0.76-fold, p<0.001) than healthy pregnancy controls. Conclusions In pregnancies complicated by severe preeclampsia, VWF:Ag and FVIII were significantly elevated, as expected. VWF:FVIII ratios also tended to be higher in preeclamptic pregnancies compared to controls. Interestingly, activity of the VWF-cleaving protease ADAMTS13 was modestly but significantly decreased in preeclampsia pregnancies, a finding for which there is conflicted literature. Three subjects with thrombocytopenia had ADAMTS13 activities >85%, indicating the thrombocytopenia was unrelated to a relative ADAMTS13 deficiency. These findings support the hypothesis that pregnancies complicated by severe preeclampsia exhibit VWF biology that is distinct from healthy pregnancy. We speculate that preeclampsia-associated VWF differences could play a direct role in the vascular pathogenesis and end-organ damage of preeclampsia. These data offer new clues towards better understanding of the mechanisms of severe preeclampsia and represent promising future targets for diagnosis and/or treatment. Disclosures: No relevant conflicts of interest to declare.

Oxidation of Key Methionine Residues in the N-Terminal Domains of ADAMTS13 Correlates with Loss of Its Proteolytic Activity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3276-3276
Author(s):  
Yi Wang ◽  
Junmei Chen ◽  
Minhua Ling ◽  
José A. López ◽  
Dominic W Chung ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Von Willebrand Factor ◽  
Hypochlorous Acid ◽  
Conflicts Of Interest ◽  
Human Neutrophils ◽  
Adamts13 Activity ◽  
Oxidative Inactivation ◽  
Rich Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3276 Deficiency of ADAMTS13 is associated with a life-threatening thrombotic microangiopathic disease—thrombotic thrombocytopenic purpura (TTP), which is characterized by the accumulation of aggregates of hyper-reactive ultra-large von Willebrand factor (VWF) and platelets in the microvasculature. In addition to TTP, systemic inflammatory syndromes such as acute systemic inflammation caused by endotoxin, acute pancreatitis, and severe sepsis demonstrate reduced ADAMTS13 activity. The cause of low ADAMTS13 activity is not known. One possible cause is oxidative inactivation of the enzyme by hypochlorous acid (HOCl), a potent oxidant released from activated neutrophils that is known to damage proteins. In this study, we exposed ADAMTS13 to HOCl produced by a myeloperoxidase (MPO)-H2O2-Cl− system and measured the proteolytic activity of oxidized ADAMTS13 using a small VWF A2 peptide and plasma VWFas substrates. ADAMTS13 activity decreased as a function of oxidant concentration. Treatment with 25 nM MPO plus 50 μM H2O2 reduced ADAMTS13 activity by over 85%. Such concentrations of MPO and H2O2 are routinely found in vivo at sites of inflammation. ADAMTS13 contains a series of structural domains: a metalloprotease domain (M), a disintegrin-like domain (D), a thrombospondin type 1 repeat (TSP1,T), a Cys-rich domain (C), a spacer domain (S), 7 additional TSP1 repeats, and 2 CUB domains. The MDTCS domains are essential for its proteolytic activity, while the C-terminal TSP1 and CUB domains may act as modulators. The MDTCS region contains 10 Met residues, Met being the amino acid residue most vulnerable to oxidation by HOCl. Using mass spectrometry, we identified 7 Met-containing peptides in this region after proteolytic digestion of ADAMTS13. Three of these 7 methionines are highly sensitive to oxidation by HOCl, including M249, M331 and M496. M249 is situated in the ”Met-turn” at the catalytic center of the metalloprotease domain. M331 and M496 are located in the disintegrin-like domain and the Cys-rich domain, respectively. The extent of oxidation of these Met residues was proportional to the HOCl concentration, and strongly correlated with loss of enzymatic activity. The same three Met residues were also oxidized after exposure of ADAMTS13 to activated human neutrophils. These observations suggest an oxidative mechanism for ADAMTS13 inactivation in systemic inflammatory syndromes and that the oxidation-sensitive Mets may serve as biomarkers for this effect. Coupled with our earlier observation that HOCl oxidation of von Willebrand factor enhances its adhesive function and renders it resistant to cleavage by ADAMTS13, these findings indicate that pathological situations during which neutrophils are activated produce extremely prothrombotic conditions, perhaps explaining why many inflammatory syndromes are associated with thrombosis in small and large blood vessels. Disclosures: No relevant conflicts of interest to declare.

An amino acid polymorphism in von Willebrand factor correlates with increased susceptibility to proteolysis by ADAMTS13

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 941-947 ◽  
Author(s):  
Derrick John Bowen ◽  
Peter William Collins
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Type I ◽  
Adamts13 Activity ◽  
Amino Acid Polymorphism ◽  
A Prospective Study ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Increased Susceptibility

Abstract The hypothesis that increased ADAMTS13 (a disintegrin and metalloprotease with thrombospondin repeats) activity or increased susceptibility of von Willebrand factor (VWF) to proteolysis by ADAMTS13 may underlie type I von Willebrand disease (VWD) in some patients was investigated. Plasma from 4 patients with type I VWD was cryoprecipitated. ADAMTS13 activity in the VWF-poor cryodepleted fraction was assessed by incubation with purified VWF; susceptibility to proteolysis of the VWF in the VWF-rich cryoprecipitate was assessed by incubation with a normal, group O cryodepleted plasma. ADAMTS13 activity was similar in all 4 type I VWD cryodepleted plasmas and comparable to a normal control plasma. In contrast, the VWF of one patient showed increased susceptibility to proteolysis by ADAMTS13. Investigation of additional family members indicated that increased susceptibility was heritable, but it did not track uniquely with type I VWD. Sequence analysis of VWF exon 28 indicated that increased susceptibility to proteolysis tracked with the “G” allele of the A/G polymorphism at position 24/1282, encoding the amino acid polymorphism Tyr/Cys1584 (“G” = Cys1584). A prospective study of 200 individuals yielded 2 Tyr/Cys1584 heterozygotes; for both, plasma VWF showed increased susceptibility to proteolysis. The finding that an amino acid polymorphism in VWF may influence susceptibility to ADAMTS13 has potentially significant implications in diverse areas. (Blood. 2004;103:941-947)

Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura

Blood ◽  
2004 ◽  
Vol 104 (7) ◽  
pp. 2081-2083 ◽  
Author(s):  
Toshihiro Uchida ◽  
Hideo Wada ◽  
Minoru Mizutani ◽  
Miho Iwashita ◽  
Hiroaki Ishihara ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Type I ◽  
Adamts13 Activity ◽  
Novel Mutations ◽  
Thrombocytopenic Purpura ◽  
Japanese Male ◽  
Old Japanese ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Von Willebrand

Abstract Congenital thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is associated with an inherited von Willebrand factor-cleaving protease (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type I domains 13]) deficiency. In this study, we identified novel mutations in the ADAMTS13 gene in a patient with TTP. The patient was a 51-year-old Japanese male who exhibited TTP symptoms at frequent intervals. The ADAMTS13 activity during acute episodes was less than 3% that of normal. The enzyme activities of the patient's father and mother were both 46%, and both parents were asymptomatic. Genetic analysis revealed that the patient was a compound heterozygote for 2 mutations. One mutation was a missense mutation in the metalloprotease domain (A250V, exon 7), and the other was a guanine to adenine substitution at the 5′ end of intron 3 (intron 3 G→A). In vitro expression studies revealed that the A250V mutation markedly reduced ADAMTS13 activity and the intron 3 G→A mutation caused abnormal mRNA synthesis. (Blood. 2004;104: 2081-2083)

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